Most bacterial pathogens have an absolute requirement for iron. The low availability of iron in most environments has led to the evolution of high affinity iron transport systems. Although iron acquisition systems have been identified in several gram negative organisms, the sources of iron used and the relative contribution of the different systems in their growth and survival in the host and in different niches in the external environment are not understood. Vibrio cholerae, the causative agent of cholera, is responsible for considerable morbidity and mortality worldwide. This organism is amenable to genetic manipulation, and several iron acquisition systems have already been identified. However, genetic analysis indicates that there are additional high-affinity iron transport systems in V. cholerae. The recent completion of its genome sequence will allow us to identify the remaining iron acquisition systems and to rigorously examine the roles of the systems in different environments and during exposure to different environmental stresses. Our first Specific Aim is to complete our characterization of V. cholerae heme transport and utilization. Our genetic data indicate that this pathogen expresses multiple heme transport systems, and we will define which genes are required for heme transport. We will also continue characterization of genes that function in the utilization of heme after it has been transported into the cell. Our second Specific Aim is to identify the transport systems used for the uptake of two exogenous siderophores used by V. cholerae, enterobactin and schizokinen. The third Specific Aim is to use our mutant collection, together with other reagents, to determine which transport systems are used during specific environmental conditions, and during growth in the vertebrate host.